This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The N-glycans were released and removed prior to b-elimination to avoid being mixed with the O-glycans in the latter's analysis. Briefly, glycoroteins were denatured by reduction and carboxyamidomethylation and then treated with Trypsin and PNGase F. Released N-linked glycans were separated from the residual peptides and O-linked glycopeptides through a C18 sep pak cartridge. Subsequently, the O-glycans were cleaved from the O-linked glycopeptides by [unreadable]- elimination procedures. After [unreadable]- elimination, released O-glycans were desalted and cleaned of borate, permethylated and analyzed by MALDI/TOF-MS and NSI-MSn. The procedures are shown in detail below. Release of N-linked glycans The dried sample was dissolved in Ambic buffer (50mM Ammonium Bicarbonate). The sample then was added with 25 mM DTT and incubated for 45 min at 50[unreadable]for reduction, followed by carboxyamidomethylation with 90mM iodoacetamide, incubated at room temperature in the dark for 45 min. The sample then was digested with the trypsin (37oC, overnight). After digestion, trypsin was inactivated by heating at 100[unreadable] C for 5 min. After cooling to room temperature, the tryptic digest was treated with PNGase F (New England BioLabs) to release the N-glycans. The tryptic/PNGase F digest then was passed through a C18 reversed phase cartridge. The carbohydrate fraction (N-linked glycans) was eluted first with 5% acetic acid and then the O-linked glycopeptides and peptides were eluted in series with 20% iso-propanol in 5% acetic acid, 40% iso-propanol in 5% acetic acid and 100% iso-propanol each into a microcentrifuge tube. The carbohydrate fraction was dried by lyophilization, whereas the other iso-propanol fractions were dried in a speed vacuum concentrator and then combined into one glass tube. Release of O-linked glycans O-linked carbohydrates were cleaved from the glycopeptides by [unreadable]-elimination procedures. Briefly, 500 [unreadable]L of 50 mM Sodiumhydroxide (NaOH) containing 19 mg of sodium borohydride was added to the sample and incubated overnight at 450C. The incubated sample then was neutralized with 10% acetic acid, desalted by passing through a packed column of DOWEXTM resins (50W x 8 [unreadable]100, Sigma Aldrich) and lyophilized. The dried sample was cleaned of borate with methanol:acetic acid (9:1) under a stream of nitrogen gas. Then the sample was passed through a C18 reversed phase cartridge to separate the O-glycans from the peptides. O-linked glycans were eluted with 5% acetic acid and lyophilized, whereas the peptides were eluted with 100% iso-propanol and dried under a stream of nitrogen gas. Preparation of the per-O-methylated carbohydrates, cleaning up by C18 The dried carbohydrate fraction was dissolved in dimethylsulfoxide and methylated with NaOH and methyl iodide (Anumula and Taylor, 1992). The reaction was quenched by the addition of water and per-O-methylated carbohydrates were extracted with dichloromethane. Per- O-methylated glycans were further cleaned of contaminants. Briefly, the glycans were loaded into a C18 sep pak cartridge and then washed with nanopure water and 15% acetonitrile. The glycans then were eluted with 85% acetonitrile. Purified glycans were dried under a stream of nitrogen gas and dissolved with methanol prior to analysis by mass spectrometry. Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI/TOF-MS) MALDI/TOF-MS was performed in the reflector positive ion mode using [unreadable]-dihyroxybenzoic acid (DHBA, 20mg/mL solution in 50%methanol: water) as a matrix. All spectra were obtained by using a 4700 Proteomics analyzer (Applied Biosystems). NanoSpray ionization-Linear Ion Trap Mass Spectrometry (NSI-LTQ/MSn) Mass spectrometric analysis was performed following the method developed at the Complex Carbohydrates Research Center (Aoki K, Perlman M, Lim JM, Cantu R, Wells L, Tiemeyer M. J Biol Chem. 2007 Mar 23;282(12):9127-42). Mass analysis was determined by using NSI-LTQ/MSn. Briefly, permethylated glycans were dissolved in 1mM NaOH in 50% methanol and infused directly into the instrument (LTQ,Thermo Finnigan) at a constant flow rate of 0.4[unreadable]L/min. The capillary temperature was set at 210o C and MS analysis was performed in the positive ion mode. The collision energy was set at 28 for MS/MS fragmentation. For total ion mapping, automated MS/MS analysis (at 28 collision energy), m/z range from 200 to 2000 was scanned in successive 2.8 mass unit windows that overlapped the preceeding window by 2 mass units.